The deformation behaviour of an Fe–19Cr–16Ni austenitic alloy was studied by micro-tensile testing of small-scale single crystals. A detailed comparison of specimens with and without nano-twins was conducted. The study revealed that the deformation behaviour depends strongly on the crystallographic orientation of the parent crystal and the spacing between nano-twin bundles. Although the yield stress in single crystals without nano-twins was governed by Schmid's law, the slip transfer inhibition by twin boundaries contributed significantly to the strengthening in nano-twinned samples. With the parent crystal oriented favourably for single-slip, the dislocations in the parent crystal glided by cross-slip onto the primary plane in the twinned crystal. For the hard-oriented parent crystal with dense nano-twins, dislocation accumulated and led to necking at an early stage. For the same orientation but with sparse nano-twins, twin growth and nucleation was promoted by dislocation reactions with the twin boundaries. These findings suggest that control of the nano-twin structure can allow the desired deformation behaviour to be tuned.